Geochemical and hydrogeological factors influencing high levels of radium contamination in groundwater in arid regions.

One of the most challenging environmental issues in arid regions is radionuclide groundwater contamination; typically, radionuclide sources, mobility, and spatial distributions are not well understood. The main objectives of this study are to investigate the groundwater hydrochemistry and identify the factors governing the radium occurrences and mobility. Groundwater samples were collected from shallow unconfined zone and deep confined Saq sandstone aquifer in the Hail area, Saudi Arabia. They were analyzed for major, minor, and trace elements as well as radium isotopes (226Ra and 228Ra). The hydrochemical relationships, water facies, spatial distribution, and the factor analysis were integrated to elucidate the governing processes in the system. The hydrochemical facies exhibited four water types characterized by earth alkaline and alkaline elements. Most samples contained sulfates and chlorides. The hydrochemical processes affecting groundwater included the dissolution of certain minerals, mixing between modern and fossil water types, and reverse ion exchange. There are high concentrations of nitrate in the unconfined zone, with low concentrations in areas under confining conditions. High radium concentrations were recorded in the groundwater, and the 226Ra and 228Ra activity concentrations of the examined samples were 11% and 98% above the World Health Organization (WHO) guidelines, respectively. The spatial distribution of 226Ra showed high activity concentration in the shallow zone under prevailing oxidizing conditions. High 228Ra contamination was identified in the confined zone where the redox potential appears to decrease and the temperature increases result in higher mobility or desorption of the radium ions. In the unconfined zone, the oxidation of Fe+2 in the groundwater and precipitation of Fe+3 in the aquifer pore spaces and co-precipitation with barite can accelerate radium adsorption. The 228Ra/226Ra ratio classified the radium groundwater enrichment into three main clusters, namely, those depending on the redox potential values, the primary source distribution, and enrichment in 226Ra relative to 228Ra. Five major factors influencing groundwater hydrochemistry were identified using factor analysis. The first factor explained the processes resulting in the dissolution of the silicate minerals and thereby increased the uranium mobility. The second factor encompassed processes leading to a rise in the groundwater salinity. The third factor identified thorium minerals as the source of the 228Ra. The fourth factor was ascribed to the decrease in radium through sorption processes or co-precipitation with barite. The fifth factor referred to by the uneven distribution of Th and U containing minerals in the aquifer.

Integrating geochemical investigations and geospatial assessment to understand the evolutionary process of hydrochemistry and groundwater quality in arid areas.

Groundwater is the key for life in arid areas. Aquifer overexploitation and climatic conditions can significantly deteriorate groundwater quality. The Al-Qassim area in central Saudi Arabia is characterized by dense agricultural use and is irrigated mainly by fossil groundwater from the Saq Aquifer. Understanding the area's hydrochemistry, major factors governing groundwater quality, and alternative uses of the groundwater are the main goals of this study. Groundwater samples were collected and examined for major, minor, and trace elements. Ionic relationships, hydrochemical facies, geospatial distributions, and multivariate analyses were conducted to assess the hydrochemical processes at play. The salinity and nitrate concentrations of the Saq Aquifer's groundwater were found to increase in the outcrop areas more than the confined areas. The spatial distributions were fragmented by three main factors: (i) modern recharge by relatively brackish water, (ii) irrigation return flow in intensive farming areas, and (iii) overexploitation and draining of deep and relatively saline zones of the aquifer. Seven water types were found representing the alkaline water with a predominance of sulfate-chloride ions and earth alkaline water with a predominance of sulfate and chloride. Mixing between fresh and brackish water, dissolution of mineral phases, silicate weathering, and reverse ion exchange were recognized as the evolutionary processes, while evaporation played a minor role. Cluster analyses characterized the fresh groundwater zone, modern groundwater recharge zone, and anthropogenic influence zone. In the confined areas, nearly all the groundwater was appropriate for domestic use and irrigation. In the outcrop areas, some limitations were found due to unsuitable conditions.

New potentially antihypertensive peptides liberated in milk during fermentation with selected lactic acid bacteria and kombucha cultures.

Compounds with the ability to inhibit angiotensin-converting enzyme (ACE) are used medically to treat human hypertension. The presence of such compounds naturally in food is potentially useful for treating the disease state. The goal of this study was to screen lactic acid bacteria, including species commonly used as dairy starter cultures, for the ability to produce new potent ACE-inhibiting peptides during milk fermentation. Strains of Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus helveticus, Lactobacillus paracasei, Lactococcus lactis, Leuconostoc mesenteroides, and Pediococcus acidilactici were tested in this study. Additionally, a symbiotic consortium of yeast and bacteria, used commercially to produce kombucha tea, was tested. Commercially sterile milk was inoculated with lactic acid bacteria strains and kombucha culture and incubated at 37°C for up to 72 h, and the liberation of ACE-inhibiting compounds during fermentation was monitored. Fermented milk was centrifuged and the supernatant (crude extract) was subjected to ultrafiltration using 3- and 10-kDa cut-off filters. Crude and ultrafiltered extracts were tested for ACE-inhibitory activity. The 10-kDa filtrate resulting from L. casei ATCC 7469 and kombucha culture fermentations (72 h) showed the highest ACE-inhibitory activity. Two-step purification of these filtrates was done using HPLC equipped with a reverse-phase column. Analysis of HPLC-purified fractions by liquid chromatography-mass spectrometry/mass spectrometry identified several new peptides with potent ACE-inhibitory activities. Some of these peptides were synthesized, and their ACE-inhibitory activities were confirmed. Use of organisms producing these unique peptides in food fermentations could contribute positively to human health.

Assessing the hydrogeochemical processes affecting groundwater pollution in arid areas using an integration of geochemical equilibrium and multivariate statistical techniques.

Rapid economic expansion poses serious problems for groundwater resources in arid areas, which typically have high rates of groundwater depletion. In this study, integration of hydrochemical investigations involving chemical and statistical analyses are conducted to assess the factors controlling hydrochemistry and potential pollution in an arid region. Fifty-four groundwater samples were collected from the Dhurma aquifer in Saudi Arabia, and twenty-one physicochemical variables were examined for each sample. Spatial patterns of salinity and nitrate were mapped using fitted variograms. The nitrate spatial distribution shows that nitrate pollution is a persistent problem affecting a wide area of the aquifer. The hydrochemical investigations and cluster analysis reveal four significant clusters of groundwater zones. Five main factors were extracted, which explain >77% of the total data variance. These factors indicated that the chemical characteristics of the groundwater were influenced by rock-water interactions and anthropogenic factors. The identified clusters and factors were validated with hydrochemical investigations. The geogenic factors include the dissolution of various minerals (calcite, aragonite, gypsum, anhydrite, halite and fluorite) and ion exchange processes. The anthropogenic factors include the impact of irrigation return flows and the application of potassium, nitrate, and phosphate fertilizers. Over time, these anthropogenic factors will most likely contribute to further declines in groundwater quality.

Spatial distribution and health risk assessment for groundwater contamination from intensive pesticide use in arid areas.

Arid and semiarid areas face major challenges in the management of scarce groundwater. This valuable resource is under pressures of population, economic expansion, contamination and over-exploitation. This research investigates groundwater vulnerability to pesticide contamination in the Al-Kharj area of Saudi Arabia. It explores the spatial distribution of pesticide concentrations in groundwater and other relevant factors. Thin permeable soils, permeable aquifers and shallow water tables, which are prevalent in the area, are especially vulnerable to pesticides. Analyses of 40 groundwater samples were performed using a gas chromatograph mass spectrometer coupled with a quadrupole mass spectrometer with a GC column. The analysis was conducted to detect 32 pesticides from different chemical families, and a total of 22 pesticides were detected. All 40 water samples were positive for at least one of the pesticides studied. In total, 21 compounds were above the quantification limit and 10 of them exceeded the legal limit. Total pesticide levels ranged from 0.18 to 2.21 µg/L, and 68 % of the analyzed samples exceeded the maximum allowable pesticide concentrations established by the European Community. Comparison of the daily intake peak (DIP) and daily intake mean (DIM) relative to the acceptable daily intake (ADI) shows that groundwater contamination with pesticides is a serious problem. Prolonged exposure to pesticides can cause adverse effects to human health and the ecosystem. Spatial distribution maps of groundwater contamination were developed using GIS. These maps will help risk managers identify vulnerable sources and provide a relative assessment of pesticide hazards to human health and the environment.

Hydrochemical modeling and assessment of groundwater contamination in northwest Sinai, Egypt.

This study aims to investigate groundwater geochemical characteristics, and to assess the effects of groundwater contamination in northwest Sinai, Egypt. A geographic information system, geochemical modeling, and statistical analyses tools were used. Twenty-five groundwater samples from a Quaternary aquifer were sampled. These water samples were analyzed for major, minor, and trace elements. The results of this study contribute to a better understanding of the hydrochemical characteristic as well as the anthropogenic processes of groundwater pollution. On the basis of these analyses, the geochemical parameters and the anomalous concentration of different elements enable the characterization of salinity sources of the brackish waters and the suspected sources of polluted water. Pollution sources are represented by waste disposal and agricultural activities as well as the probable upward leakage of highly saline water from the deeper aquifers and the saltwater intrusion. Pollution risk is high when the depth of the water table is shallow (0.3 to 15.0 m) and the aquifer has high hydraulic conductivity and poor matrix buffering capacity.

Effect of long-term topical application of dehydroepiandrosterone (DHEA) and oral estrogens on morphology, cell proliferation, procollagen A1 and androgen receptor levels in rat skin.

BACKGROUND: After cessation of estrogen secretion by the ovaries at menopause, all estrogens and almost all androgens acting in the skin of postmenopausal women are synthesized locally from dehydroepiandrosterone (DHEA), a prohormone of adrenal origin that progressively declines with age. OBJECTIVE: To better understand the effects of DHEA on the skin, ovariectomized (OVX) rats were treated for 9 months with local topical application of DHEA compared with oral conjugated equine estrogens. MATERIALS AND METHODS: Morphological evaluation, immunohistochemistry for androgen receptor (AR) and Cdc47 proliferation marker, and in situ hybridization for procollagen A1 were performed on dorsal skin. RESULTS: Local topical DHEA application increased the thickness of the granular cell layer and total epidermis in OVX animals, whereas systemic estrogens had no significant effect. Although DHEA did not affect total dermal thickness, a 190% increase in dermal procollagen A1 mRNA was observed. Moreover, DHEA treatment decreased hypodermal thickness by 47% and increased skin muscle thickness by 58%. In the epidermis, DHEA induced a non-significant increase in cell proliferation, whereas AR labeling was increased in both the epidermis and dermis by DHEA. CONCLUSIONS: Although estrogens did not significantly modify any of the above-mentioned parameters, the androgenic action of DHEA induced significant changes in all skin layers, without any sign of toxicity or lack of tolerance to DHEA after a 9-month local application of 4% (80 mg/kg) DHEA on the skin.

Androgen receptor as a potential sign of prostate cancer metastasis.

BACKGROUND: Androgen receptor (AR) expression and its modulation through the carcinogenesis process have been investigated in several studies with conflicting results. MATERIALS AND METHODS: In situ hybridization and immunocytochemistry were used to examine AR expression in prostatic needle core biopsies of benign, high grade prostatic intraepithelial neoplasia (HGPIN) and prostatic adenocarcinoma. RESULTS: A significant increase in AR mRNA levels was found in the cancerous prostatic cells when compared with the benign tissue biopsies. AR abundance in HGPIN was found to be almost half-way between that observed in benign and in cancerous tissue. In the benign prostatic epithelium, the immunocytochemistry data show that AR is exclusively expressed in the nuclei of epithelial cells. However, in 72% of examined cancer biopsies, AR was expressed in both the cytoplasm and nuclei. After examination of medical records of 100 patients diagnosed with prostate cancer, it was found that the AR was expressed in both cellular compartments of cancer cells in 81% of cases when cancer was found to have metastasized outside the prostate. In contrast, when the cancer was organ-confined, AR was localized in both the nuclei and cytoplasm in only 66% of cases. Moreover, when the AR was expressed in the cytoplasm of cancerous cells, consecutive serial sections immunostained with the mitochondrial marker suggest that AR is localized in the mitochondria. CONCLUSIONS: AR mRNA expression is significantly higher in prostate cancer when compared to benign prostatic tissue.

Expression of Patched-1 and Smoothened in testicular meiotic and post-meiotic cells.

Desert hedgehog (Dhh) signaling plays an essential role in the normal development of the testis and in the process of spermatogenesis. Little is known about the involvement in spermatogenesis of the prototypic member of the family, Ptc1, which acts to suppress hedgehog signaling through Smoothened (Smo). Here, we have examined the expression of Ptc1, Smo, and Dhh in mouse and rat seminiferous epithelium. Our findings demonstrate that Ptc1 and Smo are expressed by primary spermatocytes and by round and condensing spermatids whereas Dhh is expressed by Sertoli cells. The findings suggest that Sertoli cells coordinate Dhh-dependent spermatogenesis events via Ptc1 and Smo prior to the first meiotic division and in postmeiotic (haploid) cells, particularly during the first half of spermiogenesis.

Assessment of PARP-3 distribution in tissues of cynomolgous monkeys.

Poly(ADP-ribose) polymerase 3 (PARP-3) is a newly characterized PARP. In contrast to the two best-studied nuclear PARPs, PARP-1 and PARP-2, PARP-3 activity is apparently not stimulated by DNA damage. However, our previous work has demonstrated that PARP-3 interacts with several DNA damage response proteins, including Ku70/Ku80, DNA-PK, and PARP-1, suggesting that it contributes to the DNA damage response. Furthermore, a possible function for PARP-3 in the regulation of gene expression has been inferred from our observations that it associates with polycomb group proteins, which are responsible for epigenetic modifications leading to gene silencing. In this report, we extend our characterization of PARP-3 by revealing its distribution in the tissues and cell types of adult cynomolgous monkeys using a well-characterized PARP-3 polyclonal antibody. This study is the first to demonstrate that PARP-3 is genuinely expressed in most of the examined tissues. However, its expression is highly restricted to specific cell types of each tissue, indicating that PARP-3 expression is tightly regulated. One of the key findings of this study is that PARP-3 is highly expressed in the nuclei of epithelial cells forming the ducts of prostate, salivary glands, liver, and pancreas and in the neurons of terminal ganglia.

Effects of intravaginal dehydroepiandrosterone on vaginal histomorphology, sex steroid receptor expression and cell proliferation in the rat.

OBJECTIVE: Recent clinical studies have shown that postmenopausal hormone therapy with estrogen plus progestogen increases breast cancer risk. Moreover, intravaginal estrogen-containing pills, creams and rings lead to significant systemic exposure to estrogen, thus indicating the need for a completely novel approach to alleviate vaginal atrophy in postmenopausal women. DESIGN: We have studied the effect of intravaginal application of dehydroepiandrosterone at daily doses of 0.33 mg, 0.66 mg or 1mg in ovariectomized animals for 2 weeks, with the objective of inducing local beneficial effects in the vagina without significant systemic action. RESULTS: After 2 weeks, serum dehydroepiandrosterone, androst-5-ene-3beta,17beta-diol and dehydroepiandrosterone-sulfate were increased over a 4h time period, but serum testosterone, estradiol, estrone and dihydrotestosterone remained below detectable levels. The suppository vehicle alone produced minimal epithelial thickening limited to the vaginal distal half. The morphological effects of dehydroepiandrosterone on vaginal mucosa were observed at the lowest dose and consisted mainly of a typical androgenic effect of epithelial mucification. No change in morphological features related to cell proliferation was observed at any dehydroepiandrosterone dose on uterus, mammary gland and skin. At the highest dose, body weight showed a significant decrease, thus indicating a systemic effect on lipid accumulation. Immunohistochemistry for androgen, estrogen alpha and progesterone receptors did not reveal any significant systemic effects in the uterus, mammary gland and skin except some suggestion of increased androgen receptor labeling in mammary gland and skin at the highest dehydroepiandrosterone dose. CONCLUSION: The present data show that intravaginal dehydroepiandrosterone can exert beneficial effects limited to the vagina.

Alterations in the testis of hormone sensitive lipase-deficient mice is associated with decreased sperm counts, sperm motility, and fertility.

Hormone-sensitive lipase (HSL, Lipe, E.C.3.1.1.3) functions as a triglyceride and cholesteryl esterase, supplying fatty acids, and cholesterol to cells. Gene-targeted HSL-deficient (HSL(-/-)) mice reveal abnormal spermatids and are infertile at 24 weeks after birth. The purpose of this study was to follow the evolution of spermatid abnormalities as HSL(-/-) mice age, characterize sperm motility in older HSL(-/-) mice, and determine if mice expressing a human testicular HSL transgene (HSL(-/-)ttg) produce normal motile sperm. In situ hybridization indicated that HSL is expressed exclusively in steps 5-16 spermatids, but not in Sertoli cells. In HSL(-/-) mice, abnormalities were evident in step 16 spermatids at 5 weeks after birth, with defects progressively increasing in spermatids with age. The defects included multinucleation of spermatids, abnormal shapes and a reduction of elongating spermatids. In older HSL(-/-) mice, sperm counts appeared reduced by 42%, but this value was lower because samples were compromised by the presence of small degenerating germ cells in addition to sperm, both of which appeared of similar size and density. Sperm motility was dramatically reduced with only 11% classified as motile in HSL(-/-) mice compared to 76-78% of sperm in wild-type and HSL(-/-)ttg mice. Sperm morphology, counts, and motility were normal in HSL(-/-)ttg mice, as was their fertility. Collectively, the data indicate that HSL deficiency results in abnormal spermatid development with defects arising at 5 weeks of age and progressively increasing at later ages. HSL(-/-) mice also show a dramatic reduction in sperm counts and motility and are infertile.

Evaluation of AIbZIP and Cdc47 as markers for human prostatic diseases.

OBJECTIVES: Benign prostatic hyperplasia and prostate cancer are important public health issues. However, histologic markers for these diseases are limited. METHODS: Immunocytochemistry was used to analyze the cellular localization of AIbZIP, Cdc47, androgen receptor and estrogen receptor-beta markers. AIbZIP is a protein recently found to be more abundant in prostate cancer than in benign prostatic tissue, and Cdc47 is a cell proliferation-associated protein. The localization and modulation of androgen receptor and estrogen receptor-beta through the carcinogenesis process have been examined in several studies but controversial results were obtained. These four proteins were evaluated as potential markers of prostatic diseases in 210 needle core biopsies, including normal prostate, benign prostatic hyperplasia, low-grade and high-grade prostatic intraepithelial neoplasia, and different Gleason grades of prostatic adenocarcinoma. RESULTS: Androgen receptor and estrogen receptor-beta do not discriminate between benign and malignant specimens, while AIbZIP was able to distinguish between them. Cdc47, in contrast, discriminated not only between malignant and benign prostatic tissue, but also between benign prostatic hyperplasia and normal prostatic tissue. CONCLUSIONS: Cdc47 appears to be a sensitive marker of prostatic diseases since its expression gradually increased in parallel with the severity of the lesion. AIbZIP discriminated between benign tissue and cancer. AIbZIP and Cdc47 thus appear to be useful markers with diagnostic and prognostic values.

Epithelial trafficking of Sonic hedgehog by megalin.

We present here evidence of in vivo epithelial endocytosis and trafficking of non-lipid-modified Sonic hedgehog (ShhN) when infused into rat efferent ducts via microinjection. Initially, exogenous ShhN is detected in endocytic vesicles and early endosomes located near the apical plasma membrane of non-ciliated cells. Within 30-60 min following infusion, ShhN can be detected in lysosomes and at basolateral regions of non-ciliated cells. Basolaterally, ShhN was observed along the extracellular surfaces of interdigitated plasma membranes of adjacent cells and in the extracellular compartment underlying the efferent duct epithelium. Uptake and subcellular trafficking of infused ShhN by non-ciliated cells could be blocked by either anti-megalin IgG or the megalin antagonist, RAP. Ciliated cells, which do not express megalin, displayed little if any apical internalization of ShhN even though they were found to express Patched-1. However, ShhN was found in coated pits of lateral plasma membranes of ciliated cells as well as in underlying endocytic vesicles. We conclude that megalin-mediated endocytosis of ShhN can occur in megalin-expressing epithelia in vivo, and that the internalized ShhN can be targeted to the lysosome or transcytosed in the plane of the epithelium or across the epithelium. These findings highlight the multiple mechanisms by which megalin may influence Shh morphogen gradients in vivo.

Stage-specific expression of the Atce1/Tisp40alpha isoform of CREB3L4 in mouse spermatids.

The maturation of haploid spermatids into spermatozoa relies on the timely production of proteins required for spermatid differentiation. The mammalian CREB3L4 (cAMP responsive element binding protein 3-like 4) gene encodes a bZIP transcription factor that associates with the membrane of the endoplasmic reticulum. CREB3L4 is presumed to play an important role in protein maturation via its involvement in the cellular response to endoplasmic reticulum stress. In mice, the Creb3l4 gene gives rise to 2 distinct classes of mRNAs through the use of alternate promoters. Transcripts that initiate upstream of the first coding exon encode a 370-amino acid (aa) protein designated Tisp40beta, whereas transcripts that initiate downstream of the first coding exon encode Atce1/Tisp40alpha, a truncated (315-aa) form of Tisp40beta. In the mouse testis, Creb3l4 transcripts are known to be expressed exclusively in postmeiotic spermatids but the presence of CREB3L4 protein in spermatids has not been formally demonstrated. We produced an antibody directed against the carboxy terminus of mouse CREB3L4 and used it in immunostaining experiments to document that CREB3L4 protein accumulates in post-meiotic spermatids in a stage-specific manner. Moreover, we show that Atce1/Tisp40alpha is the major form of CREB3L4 in mouse testis. These findings suggest that testis-specific isoforms of Creb3l4 could play an important role in spermatid differentiation.

Expression of enzymes involved in estrogen metabolism in human prostate.

There is evidence that estrogens can directly modulate human prostate cell activity. It has also been shown that cultured human prostate cancer LNCaP can synthesize the active estrogen estradiol (E2). To elucidate the metabolism of estrogens in the human prostate, we have studied the expression of enzymes involved in the formation and inactivation of estrogens at the cellular level. 17beta-Hydroxysteroid dehydrogenase (17beta-HSD) types 1, 2, 4, 7, and 12, as well as aromatase mRNA and protein expressions, were studied in benign prostatic hyperplasia (BPH) specimens using in situ hybridization and immunohistochemistry. For 17beta-HSD type 4, only in situ hybridization studies were performed. Identical results were obtained with in situ hybridization and immunohistochemistry. All the enzymes studied were shown to be expressed in both epithelial and stromal cells, with the exception of 17beta-HSD types 4 and 7, which were detected only in the epithelial cells. On the basis of our previous results, showing that 3beta-HSD and 17beta-HSD type 5 are expressed in human prostate, and of the present data, it can be concluded that the human prostate expresses all the enzymes involved in the conversion of circulating dehydroepiandrosterone (DHEA) to E2. The local biosynthesis of E2 might be involved in the development and/or progression of prostate pathology such as BPH and prostate cancer through modulation of estrogen receptors, which are also expressed in epithelial and stromal cells.

Gender differences in mouse skin morphology and specific effects of sex steroids and dehydroepiandrosterone.

Sex steroids play an important role in skin morphology and physiology. To evaluate the specific effects of sex steroids, the thickness of each skin layer was measured in intact and gonadectomized (GDX) male and female mice, as well as in GDX animals treated for 3 wk with 17beta-estradiol (E2), dihydrotestosterone (DHT), or their precursor dehydroepiandrosterone (DHEA). Morphological analysis shows that the dorsal skin of intact male is thicker than in the female, whereas the epidermis and hypodermis are thicker in the female. After GDX, epidermal thickness decreases only in the female to become similar to that of the intact male. Epidermal thickness in GDX animals of both sexes increases after E2 treatment to a value similar to that of intact females, whereas an increase is observed only in females after DHEA treatment. Both DHEA and DHT increased dermal thickness whereas E2, DHT, and DHEA markedly reduced hypodermal thickness in GDX animals of both sexes. Under all conditions, the hypodermis remains thicker in females. GDX triggers a rapid hair growth from telogen to anagen with a thicker hair shaft diameter in females. This data shows that DHT, E2, and DHEA exert specific effects on the different skin layers and appendages.

Cytoplasmic localization during testicular biogenesis of the murine mRNA for Spam1 (PH-20), a protein involved in acrosomal exocytosis.

The Sperm Adhesion Molecule1 (SPAM1) is the most widely conserved sperm antigen with important roles in mammalian fertilization. Light and electron microscopy were used to localize, by in situ hybridization, the cellular and subcellular sites of Spam1 mRNA in the murine testis. Transcripts were first detected in step 3 round spermatids, gradually increased until step 8 and abruptly decreased between steps 9-11. They were predominantly localized near the ER and were not dispersed throughout the cytoplasm. Immunohistochemistry revealed that Spam1 is present on both the head and tail of sperm in the seminiferous tubules, and provided support for transcriptional regulation of its transcript. Immunocytochemistry confirmed the location of Spam1 on the tail of testicular sperm and demonstrated that it is localized to both the principal piece and the midpiece. Spam1 on epididymal sperm is localized to the midpiece of the tail and changes from a uniform distribution on the head in the caput to a regionalized pattern, first on the posterior and then on the anterior head, in caudal sperm. Spam1 on the surface of caudal sperm was shown to mediate the increase in acrosome reactions induced by the synergistic effects of HA and progesterone, as confirmed in sperm from the Rb(6.16) translocation-bearing mice which are Spam1 mutants. The similar response of human and mouse sperm to these agonists of the acrosome reaction, underscores the usefulness of the mouse as a model to study physiological aspects of SPAM1 in humans where, unlike the mouse, it is the only sperm hyaluronidase.

Spam1 (PH-20) expression in the extratesticular duct and accessory organs of the mouse: a possible role in sperm fluid reabsorption.

A widely conserved sperm antigen, the sperm adhesion molecule 1 (SPAM1 or PH-20) is a glycosylphosphatidyl inositol-linked protein with multiple roles in mammalian fertilization. It has been shown to be dually expressed in testis and epididymis and this is conserved in the four species (mouse, rat, macaques, humans) that have been studied to date. Here, we report Spam1 RNA and protein expression in the murine vas deferens and efferent ducts. In situ hybridization and immunohistochemistry indicate that transcript and protein are distributed in the nonciliated epithelial cells and that the efferent ducts have the most intense staining of all three regions of the excurrent ducts. Spam1 products were also present in the accessory organs, the prostate, and seminal vesicles and its fluid. Using hyaluronic acid substrate gel electrophoresis, hyaluronidase activity at pH 7.0 was detected in the vas deferens but was absent from the efferent ducts, the prostate, and the seminal vesicles/fluid. This suggests that Spam1 may play a nonenzymatic role in these organs. In the efferent ducts, where Spam1 is enriched in the apical (but not basolateral) membrane of nonciliated cells, it is likely to play a role in sperm concentration, which is the established function of that organ.

The kinesin KIF17b and RNA-binding protein TB-RBP transport specific cAMP-responsive element modulator-regulated mRNAs in male germ cells.

Testis brain RNA-binding protein (TB-RBP), the mouse orthologue of the human protein Translin, is a widely expressed and highly conserved protein with proposed functions in chromosomal translocations, mitotic cell division, and mRNA transport, stabilization, and storage. Targeted inactivation of TB-RBP leads to abnormalities in fertility and behavior. A testis-enriched kinesin KIF17b coimmunoprecipitates with TB-RBP in a RNA-protein complex containing specific cAMP-responsive element modulator (CREM)-regulated mRNAs. The specificity of this interaction is confirmed by in vivo RNA-protein crosslinking and transfections of hippocampal neurons. Combining in situ hybridization and immunohistochemistry at the electron microscope level, a temporally sequential dissociation of KIF17b and TB-RBP from specific mRNAs is detected with TB-RBP release coincident with the time of mRNA translation, indicating a separation of the processes of transport and translation. We conclude that KIF17b serves as a molecular motor component of a TB-RBP-mouse ribonucleoprotein complex transporting a group of specific CREM-regulated mRNAs in mammalian male postmeiotic germ cells. Because KIF17b has been reported to control CREM-dependent transcription in male germ cells by regulating the intracellular location of the transcriptional coactivator activator of CREM in testis, this indicates that one kinesin links the processes of transcription and transport of specific mRNAs in mammalian male germ cells.